Tn554 is a transposable element in Staphylococcus aureus that encodes resistances to spectinomycin and to the macrolide-lincosamide-streptogramin B antibiotics. Tn554 inserts efficiently into a unique site in the S. aureus chromosome. Sequence analysis of the Tn554 insertion junctions revealed that transposition does not generate a duplication of any target sequences, nor do the termini contain repeated sequences, suggesting that Tn554 may transpose by a mechanism more similar to prophage insertion that to other transposable elements. The nucleotide sequence of Tn554 contains six open reading frames; two are the antibiotic resistance markers ermA and spc; three, tnpA, tnpB and tnpC, are required for transposition; the sixth is unidentified. The tnpA and tnpB products have been identified in an in vitro transcription-translation system from Bacillus subtilis. A major goal of this proposal is to identify, isolate and characterize the transposition bunctions, with special emphasis on those that are involved in the specific recognition and recombination with the unique insertion site. A classic genetic approach will be used. Point mutations in the target site will be obtained by in vitro mutagenesis techniques. These will be sequenced and the base changes correlated with the efficiency with which each mutant acts as a target for Tn554 transposition. Tn554 mutants with altered site-specificity, but with wild type transposition properties, will then be isolated by selection for transposition into the mutation insertion sites, in a host in which the wild type chromsomal insertion site has been deleted. Mutants that lack any residual specificity will also be selected. The mutants will be mapped by complementation with transposition-defective mutations and by nucleotide sequencing. A knowledge of the nucleotides essential for the target to be funcitonal, and of the positions and types of mutations in Tn554-encoded proteins that alter the affinity for the target DNA, will allow the formulation of a model for the interaction of Tn554 with the insertion site. The proteins specified by Tn554 will be isolated from E. coli carrying clones containing the appropriate Tn554 sequences. A site-specific endonuclease assay will be used to monitor the purifications where possible; otherwise the proteins will first be isolated as fusions with Beta-galactosidase and antibody against the fusion proteins will be used to isolate the native proteins using affinity chromatography. These proteins will be hcaracterized with respect to specific DNA binding and possible nuclease and topoisomerase activities.